| Background:Depression is a chronic recurring illness that results in major social and economic consequences. Clinical symptoms like low self-esteem, loss of interest or pleasure, fatigue, and feelings of worthlessness are characteristic of depression. Recent research suggests that a worldwide prevalence of depression was approximately17%. An epidemiological survey carried out from 2001 to 2005, of 113 million adults from four provinces in China demonstrated a 6% prevalence rate for depression. About 25-30% of patients with depression have suicidal behavior, the frequency of suicide is 11-19%. WHO’s Global Burden of Disease Cooperative Research estimated depression will become the second largest disease burden in 2020 in the world Following coronary heart disease. However, the current mechanisms of pathogenesis and treatment of depression remains unclear.In the past few decades, the monoaminergic systems (serotonin and norepinephrine) have received the most attention in the neurobiology and treatment of depression. To date, it has been recognized that monoamine deficits are not solely sufficient to explain the pathophysiology of depression and the mechanism of action of antidepressants. Moreover, increasing evidence indicates an important role for the glutamatergic system in fundamental processes related to the occurrence and treatment of depression.At present, a lots of studies recognized that stress plays an important role in the pathogenesis of depression. The primary role of stress has been attributed to glucocorticoid hormones, major mediators in the stress response. Accordingly, it has been argued that stress-induced rise of glucocorticoid hormones and binding to synaptic MR/GR, in turn, cause massive release of glutamate from glutamate neuron into the synaptic cleft, resulting in a sharp increase in the synaptic cleft. Studies have shown that excess glutamate in synaptic cleft produces neurotoxicity to damage structure and function of glial cells and neurons, which is related to the occurrence with some neuropsychiatric disorders (including depression). Due to the lack of specificity of metabolic enzymes in the synaptic cleft, the excess glutamate is to clear primarily through the glutamate transporter presenting on astrocyte cells.To date, five subtypes of glutamate transporters have been cloned:EAAT1 (rodent nomenclature GLAST:glutamate aspartate transporter), EAAT2 (rodent nomenclature GLT-1:glutamate transporter 1), EAAT3 (rodent nomenclature EAAC1:excitatory amino acid carrier 1), EAAT4, and EAAT5. GLAST and GLT-1 are expressed in glial cells, whereas EAAC1 and EAAT4 are expressed in neurons. EAAT5 is present in the glia and neurons of the retina. GLT-1 is expressed at high levels at the astrocytic membrane surface in the hippocampus and the prefrontal cortex (PFC) and is involved in over 90% of total glutamate uptake. GLT-1 transports glutamate into astrocytes, where glutamate is converted to nontoxic glutamine by the enzyme glutamine synthetase (GS).Data from patients with depression and animal models of depression have revealed alterations in the levels of expression of GLT-1. However, experimental data on brain GLT-1 levels are, to some extent, inconsistent in human post mortem tissue and animal studies. These discrepancies imply that the role of GLT-1 in the pathophysiology of depression and the action of antidepressants remain obscure. The relationship between GLT-1 and depression requires further study.Fluoxetine (FLX), a selective serotonin (5-HT) reuptake inhibitor, is one of the most commonly prescribed antidepressants. Fluoxetine exert their initial pharmacological effects by increasing the extracellular levels of serotonin in the synaptic cleft. Nevertheless, patients with depression must take regular doses for at least 3 to 4 weeks before they are likely to experience a full therapeutic effect. The inconsistencies suggest that other mechanisms in addition to the monoamine neurotransmitter systems are possibly related to antidepressant activity of fluoxetine.There are studies that the glutamatergic system is associated with antidepressant activities of fluoxetine. However, it had not been reported at home and abroad whether the glutamate transporter GLT-1 is involved in the antidepressant activities of fluoxetine.Objective:To investigate the effects of CUS stress and fluoxetine on the changes of protein levels of GLT-1 of astrocyte in hippocampus and prefrontal cortex in rat depression model. And to further explore the possible molecular mechanism of the pathogenesis and treatment of depression.Method:(1) Sixty male Sprague-Dawley (SD) rats were randomly separated into three groups:the control+saline (Control+S; n=20), CUS+saline (CUS+S; n=20), and CUS+ fluoxetine (CUS+FLX; n=20) groups. The sucrose preference and open field tests were conducted after CUS and FLX treatment. The rats were housed in groups during the experimental period, but were stressed, injected and underwent behavioral assessments in individual housing. All rats were decapitated immediately after the last behavioral experiment. Ten rats in each group were used for the expression of GLT-1 by immunohistochemistry. An additional ten rats in each group were used for GLT-1 western blotting analysis. (2) Chronic Unpredictable Stress (CUS):The rats were subjected to various stressors for 5 weeks (2 stressors per day). Ten stressors were used in the experiment:immobilization for 2 h (in a 25 cm×8 cm cylindrical plastic rodent restrainer), nip trail for 1 min, swimming in 4℃ cold water for 5 min, swimming in 45℃ warm water for 5 min, cage tilting (45°) for 24 h, damp sawdust (200 ml water in a cage) for 24 h, shaking for 15 min (120 rpm rocking bed), food deprivation for 24 h, water deprivation for 24 h, and alterations of the light-dark cycle. The rats were exposed to the stressors individually. The same stressor was not administered to any individual for two consecutive stages. The rats in the Control+S group were not subjected to stressors. (3) Behavioral test:(ⅰ) Sucrose preference test:Before each test, the rats were deprived of water for 12 h (8:00 p.m. to 8:00 a.m.). The rats were allowed to drink a bottle of 1% sucrose solution and water for 1 h. The total consumption of the sucrose solution and water was measured by comparing the bottle weight before and after the test. The sucrose preference was calculated by the following formula:the sucrose preference (%)= (total sucrose solution consumption/total water+ total sucrose solution consumption) x 100%. (ⅱ) Open field test:Each animal was placed at the center of a dimly illuminated rectangular cage (120 cm×90 cm×35 cm) and observed for 10 min continuously by a camera, which was used to assess spontaneous activity. The total traveled distance, moved velocity, and frequencies of rearing (standing upright on the hind legs while the forepaws are free) were automatically calculated with a video tracking system (Ethovision 3.0; Noldus, the Netherlands). The cage was cleaned thoroughly before the next test. (ⅲ) FLX treatment:FLX was dissolved in saline immediately prior to administration. The rats received a daily intraperitoneal injection of FLX (10 mg/kg) or an equal volume of saline vehicle for 4 weeks. (ⅳ) After the last bahvior test, rats were sacrificed, hippocampus and prefrontal cortex were isolated. Changes of glutamate transporter GLT-1 expression in hippocampus and prefrontal cortex were detected by using immunohistochemistry method. (ⅴ) The expression of GLT-1 in hippocampus and prefrontal cortex were analyzed used westen blot. (ⅵ) Statistics:Data analysis was performed using SPSS version 17 software. The results are expressed as the mean±S.D. Data were analyzed by one-way ANOVA followed by the Bonferroni test for post hoc multiple range comparisons. The statistically significant group difference was set asp< 0.01.Results:(1) Compared with the non-stressed controls, the percentage of sucrose consumption, the total traveled distance, the moved velocity, and the frequencies of rearing in the stressed animals were significantly decreased (p<0.01). The sucrose preference and activity in the open field test were no differences between the controls and the animals treated with FLX for 4 weeks. In contrast, there were significantly different between the CUS+S group and the other groups (p<0.01 of all). (2) Using immunohistochemistry, the control rats showed a moderate immunoreactivity for GLT-1 in the CA1, CA3, and DG regions of the hippocampus and prefrontal cortex, whereas the samples from the stressed rats revealed a weak immunoreactivity. However, the samples from the FLX-treated rats displayed a moderate immunoreactivity for GLT-1. The immunoreactivity for GLT-1 of the FLX-treated rats was significantly upregulated compared with the stressed rats and was similar to the samples from the control rats. Using quantitative representation of the expression of GLT-1 by integrated optical density (IOD), we found significant reductions of GLT-1 in stress rats in the hippocampal CA1 regions (versus the control rats:p<0.01, versus the FLX-treated rats: p<0.01), CA3 regions (versus the control rats:p<0.01, versus the FLX-treated rats: p<0.01), DG regions (versus the control rats:p<0.01, versus the FLX-treated rats: p<0.01) and prefrontal cortex(versus the control rats:p<0.01, versus the FLX-treated rats:p<0.01). (3) Using Western Blot Analysis, there were significant reductions of GLT-1 in the stressed rats in the hippocampus and prefrontal cortex (versus the control rats:p<0.01, versus the FLX-treated rats:p<0.01). Compared with the control rats, we found no significant differences in GLT-1 expression in FLX-treated rats in the hippocampal regions and prefrontal cortex.Conclusion:Our results suggest that there were a significant behavioral changes and a significant decrease of protein levels of GLT-1 of astrocyte in the hippocampus and prefrontal cortex in the depression model rats. Concurrently, the rats treated with FLX for four weeks reversed stress-induced behavioral changes and up-regulated the GLT-1 levels to approximately the same level as the non-stressed control animals. Moreover, our results suggest that GLT-1 may be involved in FLX antidepressant activities. Taken together, our studies further support the notion that GLT-1 represents an attractive candidate molecule associated with the fundamental processes of depression and may be a potentially novel, pharmacological target for the treatment of depression. |
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